Proper mitochondrial function requires the coordinated expression of 37 genes encoded in the circular genome inside the mitochondrion, and over 1000 genes encoded on nuclear chromosomes. This intracellular, intergenomic communication presents a complicated network of interacting genes that are critical for the energy production that sustains life. Because each of these genes is variable in natural populations, each gene-by-gene interaction can be altered by the variation among individuals. Moreover, the mitochondrion is a hub of many signaling pathways that sense nutrients, oxygen, redox state of the cell, and temperature making it sensitive to environmental conditions. As a result, these interactions present a complex system that lies between genotype and phenotype. The first Aim is to dissect this mitochondrial-nuclear (mitonuclear) interaction by generating and phenotyping all pairs of genotypes from 40 well-characterized inbred strains of Drosophila (the Drosophila Genetics Reference Panel), and 6 sequenced mtDNAs from D. melanogaster and D. simulans. This will add an important mtDNA component to the DGRP resource. This Aim will quantify metabolite profiles and resistance to hypoxic stress in alternative dietary and oxygen environments, respectively. This will test the hypothesis that environmental stress alters the epistatic component to mitonuclear interactions and further test the hypothesis that mtDNA disease states are more common in males, which do not transmit mtDNA. The second Aim will identify specific nuclear transcripts whose expression are altered by mtDNA background and these same environmental stressors (diet composition or oxygen tension). This will test the hypothesis that genes in the pathways of central nutrient metabolism and hypoxia signaling are uniquely sensitive to mtDNA genotype. In both Aims, the genealogy of the mtDNAs used will be used in a neutrality test of mtDNA-phenotype association that allows partitioning of traits to distinct classes of mutations in the mtDNA haplotypes. Because most QTL and genome wide association studies (GWAS) do not test for mtDNA or joint mitonuclear interaction effects on phenotype, the proposed experimental design will addresses this shortcoming directly, and may identify components of 'missing heritability' not identified in GWAS. The research will also contribute to knowledge of specific pathways relevant to mitochondrial function that may lead to pharmaceutical treatments. By manipulating diet or oxygen levels in the environment, we may identify novel roles for mitonuclear interactions affecting obesity and sensitivity to hypoxic stress.